A growing number of neurosecretory peptides are being found to be derived from a polyhormonal precursor which contains within its sequence the structures of more than one secretory product. This raises fundamental questions concerning the biochemical mechanisms controlling the production of these peptides and their routing to appropriate subcellular locations. Specifically, to what extent do these functions depend on the structure of the precursor, as opposed to properties of the processing enzymes? This question will be addressed in experiments on the processing sequence leading to the neurosecretory egg-laying hormone (ELH) in Aplysia bag cells. The precursor in this sequence is of known primary structure and gives rises to ELH and other secretory peptides by a proteolytic processing sequence which has been characterized in detail. Moreover, intermediates in this sequence which lead to different products are routed to different locations within the cell. There are a number of putative cleavage sites in the precursor, but which of these are actually used is not yet known. Moreover, the precursor and its intermediates are probably membrane-bound, but the regions of these molecules mediating this association, which may be important in routing, have yet to be determined. In addition, it is not yet known if the precursor and intermediates are cleaved by the same or different endoproteases. The experiments in this proposal are designed to answer these questions. Partial sequence analysis of the precursor, intermediates, and products will be used to identify cleavage sites by reference to the known structure of the precursor. Comparison of peptide maps of the precursor synthesized in a cell-free translation system to those from the precursor synthesized by intact cells will identify the region containing the "signal" sequence. Labelling with a lipid-soluble photoactivated probe will yield information on regions of membrane association. Characterization of precursor and intermediate cleaving activity in bag cell homogenates should reveal the degree of multiplicity of the processing endoproteases, and subcellular fractionation techniques will be used to determine their subcellular distribution. These experiments should yield insights into the fundamental cellular processes controlling the production of neuropeptides and peptide hormones.